Methods for identifying nucleic acid ligands

ABSTRACT

The present invention generally relates to methods for identifying nucleic acid ligands of a target molecule. In certain embodiments, the invention provides methods for identifying a nucleic acid ligand of a target molecule from a candidate mixture of nucleic acids, including contacting at least one target molecule with a candidate mixture of nucleic acids, in which the nucleic acids have different affinities for the target molecule, and separating in a single step nucleic acids that bind the target molecule with greatest affinity from nucleic acids that bind the target molecule with a lesser affinity and nucleic acids that do not bind the target molecule, thereby identifying the nucleic acid ligand of the target molecule.

FIELD OF THE INVENTION

The present invention generally relates to methods for identifyingnucleic acid ligands of a target molecule.

BACKGROUND

A nucleic acid ligand (aptamer) is a nucleic acid macromolecule (e.g.,DNA or RNA) that binds tightly to a specific molecular target Like allnucleic acids, a particular nucleic acid ligand may be described by alinear sequence of nucleotides (A, U, T, C and G), typically 15-40nucleotides long. In solution, the chain of nucleotides formsintramolecular interactions that fold the molecule into a complexthree-dimensional shape. The shape of the nucleic acid ligand allows itto bind tightly against the surface of its target molecule. In additionto exhibiting remarkable specificity, nucleic acid ligands generallybind their targets with very high affinity, e.g., the majority ofanti-protein nucleic acid ligands have equilibrium dissociationconstants in the picomolar to low nanomolar range.

Nucleic acid ligands are generally discovered using an in vitroselection process referred to as SELEX (Systematic Evolution of Ligandsby EXponential enrichment). See for example Gold et al. (U.S. Pat. No.5,270,163). SELEX is an iterative process used to identify a nucleicacid ligand to a chosen molecular target from a large pool of nucleicacids. The process relies on standard molecular biological techniques,using multiple rounds of selection, partitioning, and amplification ofnucleic acid ligands to resolve the nucleic acid ligands with thehighest affinity for a target molecule.

While successful at eventually generating high affinity nucleic acidligands, the SELEX process requires multiple time consuming rounds ofselection, partitioning, and amplification, because during nucleic acidligand selection, low affinity nucleic acid ligands are at an increasedconcentration in a nucleic acid ligand library compared to high affinitynucleic acid ligands. SELEX requires multiple rounds to isolate the highaffinity nucleic acid ligands because the low affinity nucleic acidligands must be eliminated gradually to ensure eventual selection of thehigh affinity nucleic acid ligands.

There is an unmet need for methods that can more efficiently discovernucleic acid ligands to target molecules.

SUMMARY

The present invention provides methods for rapid (e.g., single step) anddirect isolation of nucleic acid ligands of high affinity to a targetmolecule. Methods of the invention accomplish single step identificationof nucleic acid ligands by employing selective separation protocols(e.g., gel electrophoresis or HPLC gradient elution) that eliminateundesirable competition for the target molecule among nucleic acids thatbind the target molecule with greatest affinity, nucleic acids that bindthe target molecule with a lesser affinity, and nucleic acids that donot bind the target molecule. The selective separation protocolsgenerate conditions in which the nucleic acids that bind the targetmolecule with a lesser affinity and nucleic acids that do not bind thetarget molecule cannot form complexes with the target molecule or canonly form complexes with the target molecule for a short period of time.In contrast, the conditions of the separation protocols allow nucleicacids that bind the target molecule with greatest affinity to formcomplexes with the target molecule and/or bind the target molecule forthe greatest period of time, thereby separating in a single step thenucleic acids with the greatest affinity for the target molecule, i.e.,the nucleic acid ligands, from the remainder of a candidate mixture ofnucleic acids.

An aspect of the invention provides methods for identifying a nucleicacid ligand of a target molecule from a candidate mixture of nucleicacids including contacting at least one target molecule with a candidatemixture of nucleic acids, in which the nucleic acids have differentaffinities for the target molecule, and separating in a single stepnucleic acids that bind the target molecule with greatest affinity fromnucleic acids that bind the target molecule with a lesser affinity andnucleic acids that do not bind the target molecule, thereby identifyingthe nucleic acid ligand of the target molecule. The target molecule canby any type of biomolecule or a complex of biomolecules. Exemplarytarget molecules include a cell, cellular fragment, protein or portionthereof, an enzyme, a peptide, an enzyme inhibitor, a hormone, acarbohydrate, a glycoprotein, a lipid, a phospholipid, and a nucleicacid. In a particular embodiment, the target molecule is an infectiousprion.

Separating can be accomplished by any of numerous methods that providefor selective single step separation of nucleic acids that bind thetarget molecule with greatest affinity from nucleic acids that bind thetarget molecule with a lesser affinity and nucleic acids that do notbind the target molecule. In certain embodiments, separating includesloading the target molecule into a gradient gel, applying an electriccurrent to cause the target molecule to migrate to a position in thegel, in which the target molecule remains immobilized at that positionin the gel, loading the candidate mixture into the gel, and applying anelectric current to cause the candidate mixture to migrate through thegel, in which the nucleic acids with the greatest affinity for thetarget molecule (i.e., nucleic acid ligands) bind to the target moleculeimmobilized in the gel, and the nucleic acids with lesser affinity forthe target molecule and nucleic acids with no affinity for the targetmolecule migrate to an end of the gel. The nucleic acid ligand/targetmolecule complex is then cut from the gel, and the nucleic acid ligandsare then dissociated from the target molecules using a chaotropic agent.

In other embodiments, separating includes incubating the candidatemixture of nucleic acids with a plurality of target molecules to formnucleic acid/target molecule complexes, in which the target moleculesare bound to beads, and eluting the nucleic acids from the complexesthat have been loaded onto an HPLC column by applying an HPLC gradientprofile, in which nucleic acids with the greatest affinity for thetarget molecule elute at an end portion of the gradient profile and thenucleic acids with a lesser affinity for the target molecule and nucleicacids with no affinity for the target molecule elute prior to the endportion of the gradient profile. Many different HPLC gradient elutionprofiles are known in the art. An exemplary HPLC gradient elutionprofile may include a linear increasing concentration of the targetmolecule, in which an end portion of the gradient profile may include alinear increasing concentration of the target molecule and a chaotropicagent (e.g., urea, guanidinium chloride, SCN⁻, or LiBr). Prior toincubating, the method may further include loading the target moleculesbound to the beads into an HPLC column. Alternatively, subsequent toincubating, the method may further include loading the candidate mixtureand the nucleic acid/target molecule complexes onto an HPLC column.

Methods of the invention may further include sequencing the nucleic acidligand. Sequencing may be accomplished by any method known in the art.In a particular embodiment, sequencing is a single-molecule sequencingby synthesis technique. The nucleic acid ligand may include DNA or RNA.

Another aspect of the invention provides methods for identifying anucleic acid ligand of a target molecule from a candidate mixture ofnucleic acids including contacting a candidate mixture of nucleic acidsto a target molecule under conditions to form a plurality oftarget/nucleic acid complexes, in which the nucleic acids have differentaffinities for the target molecule and the nucleic acids that form thecomplex are the nucleic acids that have an increased affinity for thetarget molecule compared to the remainder of the nucleic acids in themixture, separating the target/nucleic acid complexes from the remainderof the mixture, and dissociating the complexes in a manner in whichbound nucleic acids dissociate from the target molecules at differentrates based upon the different affinities of the bound nucleic acids tothe target molecule, in which nucleic acids that dissociate from thetarget molecule at slowest rate are identified as the nucleic acidligands of the target molecule. The method can further includecollecting the nucleic acid ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic diagram showing steps for a single stepseparation protocol using HPLC gradient elution from an HPLC column.

FIG. 2 is a graph showing a ratio of A₂₆₀/A₂₈₀ during nucleic acidligand elution from an HPLC column as a function of eluted volume.

FIG. 3 panels A and B are graphs showing absorbance rates as a functionof the rPA amount. Panel A is 0.08 to 3.2 μg rPA per well. Panel B is0.08 to 5.4 μg rPA per well.

FIGS. 4A-B are photographs of gels containing isolated nucleic acidligands bound to infectious prions. FIG. 4A shows a DNA titration gelshowing band density as function of DNA oligomer concentration. FIG. 4Bshows a gel obtained by sequential application and electrophoresis ofprotein and DNA randomer solutions. The area of PrP^(Sc) protein stainedwith EB due to presence of DNA is circled.

FIG. 5 is a photograph of a gel containing the extracted DNA nucleicacid ligands. Separation was obtained by PAGE electrophoresis.

FIG. 6 is a photograph of a gel showing PCR product. Lane A representsamplified starting DNA oligomers and Lane B represents amplified prionspecific oligomers.

FIG. 7 is a graph showing results of RT-PCR of nucleic acid ligands thatwere dissociated from PrP^(CWD).

FIG. 8 is a graph showing nucleic acid ligand detection of PrP^(CWD)using DNAse I treatment to remove unbound nucleic acid ligands.

FIGS. 9A-B show confirmation of specific PCR product after amplificationof the nucleic acid ligands. FIG. 9A is a graph showing a melt curveanalysis. FIG. 9B shows an agarose gel electrophoresis.

DETAILED DESCRIPTION

The present invention generally relates to methods of identifyingnucleic acid ligands (aptamers) of a target molecule. An aspect of theinvention provides methods for identifying a nucleic acid ligand of atarget molecule from a candidate mixture of nucleic acids. A candidatemixture is a mixture of nucleic acids of differing sequence, from whichto select a desired ligand. The source of a candidate mixture can befrom naturally-occurring nucleic acids or fragments thereof, chemicallysynthesized nucleic acids, enzymically synthesized nucleic acids ornucleic acids made by a combination of the foregoing techniques. Anucleic acid includes DNA or RNA, single-stranded or double-stranded andany chemical modifications thereof. Such modifications include, but arenot limited to, modifications at cytosine exocyclic amines, substitutionof 5-bromo-uracil, backbone modifications, methylations, unusualbase-pairing combinations and the like. Protocols for designingcandidate mixtures and make-up of candidate mixtures are shown in, forexample, Griffin et al. (U.S. Pat. No. 5,756,291) and Gold et al. (U.S.Pat. No. 5,270,163), the contents of each of which are incorporated byreference herein in their entirety.

A target molecule refers to a compound of interest for which a ligand isdesired. A target molecule includes a biomolecule that could be thefocus of a therapeutic drug strategy or diagnostic assay, including,without limitation, proteins or portions thereof, enzymes, peptides,enzyme inhibitors, hormones, carbohydrates, glycoproteins, lipids,phospholipids, nucleic acids, and generally, any biomolecule capable ofturning a biochemical pathway on or off or modulating it, or which isinvolved in a biological response. Targets may be free in solution orassociated with cells or viruses, as in receptors or envelope proteins.Any ligand which is of sufficient size to be specifically recognized byan oligonucleotide sequence can be used as the target. Thus,glycoproteins, proteins, carbohydrates, membrane structures, receptors,organelles, and the like can be used as the complexation targets.

Target molecules that are not conventionally considered to bebiomolecules are also appropriate for the methods described herein.Examples of non-biomolecule targets include intermediates orend-products generated by chemical synthesis of compounds used intherapeutic, manufacturing or cosmetic applications, including polymersurfaces, such as those useful in medical applications. Nucleic acidligands may be used to specifically bind to most organic compounds andare suitably used for isolation or detection of such compounds.

A target molecule also includes intracellular, extracellular, and cellsurface proteins, peptides, glycoproteins, carbohydrates, includingglycosaminoglycans, lipids, including glycolipids and certainoligonucleotides. An exemplary list of target molecules for which thenucleic acids of the invention may be prepared is shown in Griffin etal. (U.S. Pat. No. 5,756,291).

In particular embodiments, the target molecule is an infectious prion. Aprion is an infectious agent that is composed of protein that propagatesby transmitting a mis-folded protein state. Prions causeneurodegenerative disease by aggregating extracellularly within thecentral nervous system to form plaques known as amyloid, which disruptthe normal tissue structure. This disruption is characterized by holesin the tissue with resultant spongy architecture due to the vacuoleformation in the neurons. Other histological changes includeastrogliosis and the absence of an inflammatory reaction. While theincubation period for prion diseases is generally quite long, oncesymptoms appear the disease progresses rapidly, leading to brain damageand death. Neurodegenerative symptoms can include convulsions, dementia,ataxia (balance and coordination dysfunction), and behavioral orpersonality changes.

The protein that prions are made of (PrP) is found throughout mammals,such as humans, sheep, cow, pigs, goats, etc. However, PrP found ininfectious material has a different structure and is resistant toproteases, enzymes that normally break down proteins. The normal form ofthe protein is named PrP, while the infectious form is named PrP^(Sc).PrP ^(C) is a normal protein found on the membranes of cells, having 209amino acids (in humans), a disulfide bond, a molecular weight of 35-36kDa, and a mainly a-helical structure. Several topological forms exist,one cell surface form anchored via glycolipid and two transmembraneforms. PrP ^(C) is readily digested by proteinase K and can be liberatedfrom the cell surface in vitro by the enzyme phosphoinositidephospholipase C (PI-PLC), which cleaves the glycophosphatidylinositol(GPI) glycolipid anchor. PrP^(C) may function in cell-cell adhesion ofneural cells, and/or be involved in cell-cell signaling in the brain.

The infectious isoform of PrP, known as PrP^(Sc), is able to convertnormal PrP^(C) proteins into the infectious isoform by changing theconformation, which alters the way the proteins interconnect. PrP^(Sc)has a higher proportion of β-sheet structure in place of the normala-helix structure. Aggregations of these abnormal isoforms form highlystructured amyloid fibers, which accumulate to form plaques. The end ofeach fiber acts as a template onto which free protein molecules mayattach, allowing the fiber to grow. Only PrP molecules with an identicalamino acid sequence to the infectious PrP^(Sc) are incorporated into thegrowing fiber.

Methods of the invention involve contacting at least one target moleculewith a candidate mixture of nucleic acids, in which the nucleic acidshave different affinities for the target molecule. The nucleic acidligands in the candidate mixture have specific binding regions that arecapable of forming complexes of the greatest affinity with an intendedtarget molecule in a sample in which remaining nucleic acids in thecandidate mixture either do not form a complex with the target moleculeor form a complex with the target molecule with a lesser affinity thanthe nucleic acid ligands.

Specificity of binding is measured in terms of the comparativedissociation constants (Kd) of the nucleic acid ligands for target ascompared to the dissociation constant with respect to the nucleic acidligands and other nucleic acids in the candidate mixture. Typically, theKd for the nucleic acid ligand with respect to the target molecule willbe 2-fold, 5-fold, or 10-fold less than the Kd with respect to targetand the remaining nucleic acids in the candidate mixture. In certainembodiments, the Kd will be 50-fold less, 100-fold less, or 200-foldless. The binding affinity of the nucleic acid ligands with respect tothe target molecule is measured in terms of Kd. The value of thisdissociation constant can be determined directly by well-known methods,and can be computed for complex mixtures by methods such as those shownin Caceci et al. (Byte, 9:340-362, 1984).

Methods of the invention further include separating in a single stepnucleic acids that bind the target molecule with greatest affinity fromnucleic acids that bind the target molecule with a lesser affinity andnucleic acids that do not bind the target molecule, thereby identifyingthe nucleic acid ligand of the target molecule. The selective separationprotocols generate conditions in which the nucleic acids that bind thetarget molecule with a lesser affinity and nucleic acids that do notbind the target molecule cannot form complexes with the target moleculeor can only form complexes with the target molecule for a short periodof time. In contrast, the conditions of the separation protocols allownucleic acids that bind the target molecule with greatest affinity toform complexes with the target molecule and/or bind the target moleculefor the greatest period of time, thereby separating in a single step thenucleic acids with the greatest affinity for the target molecule, i.e.,the nucleic acid ligands, from the remaining nucleic acids in thecandidate mixture.

Separating can be accomplished by any of numerous methods that providefor selective single step separation of nucleic acids that bind thetarget molecule with greatest affinity from nucleic acids that bind thetarget molecule with a lesser affinity and nucleic acids that do notbind the target molecule. Exemplary separating procedures include HPLCgradient elution and gel electrophoresis.

FIG. 1 is a schematic diagram showing steps for a single step separationprotocol using HPLC gradient elution from an HPLC column. In thisseparation protocol, a competition of epitopes for nucleic acid ligandsis generated such that at a certain ratio of target to nucleic acidligand concentration, almost all nucleic acid ligands exhibitingapparent affinity to the target molecule are bound to the target, whichis provided in excess of the nucleic acids in the candidate mixture.When binding is completed, the system is exposed to a flow of freshsolution of gradually decreasing target concentration. During gradualdecrease of target concentration, different equilibriums (the equationfor which is shown in Equation 1 below), for each given nucleic acid /target complex is achieved.A+T→AT K ^(i) _(D) =[A]:[T]·[AT] ⁻¹   Equation 1where: A is an aptamer, T is a target molecule, AT is an aptamer/targetmolecule complex, and K^(i) _(D) is the dissociation constant for agiven aptamer/target molecule complex.

Because K^(i) _(D) does not depend on concentrations of A, [A], and T,[T], and based on the law of mass action, gradual increase of [T]results in decrease of concentration of a given aptamer [A_(i)]. Thus,during the elution process the effluent will be enriched in aptamers ofhigher affinity to target, and eventually the final fractions containthe aptamers of the highest affinity to the target. It is envisionedthat some sequences may show exceptionally high affinity to target andwill not be apparently eluted even when the target in the solutionreaches its maximum. To obtain those very selective structures the endof the elution process may include increasing concentration of achaotropic agent, such as urea, guanidinium chloride, SCN⁻, or LiBr.Thus the fractions of the highest affinity aptamers will be elutedgradually.

FIG. 1 shows that beads are saturated with target molecule. The beadscan be any beads suitable for use during HPLC protocols. Numerous typesof beads are known in the art and are commercially available, forexample, from Sigma-Aldrich (St. Louis, Mo.). The beads can be porousbeads or nonporous beads. FIG. 1 shows the beads as nonporous beads. Thebeads are activated using procedures known in the art and then incubatedwith target molecule, thereby allowing the target molecules to bind tothe beads. An exemplary protocol for activating HPLC beads and bindingtarget molecules to the beads in shown in Example 1 below. Incubationtimes can be easily determined by one of skill in the art. Factors thatinfluence incubation time include type of target molecule, type of bead,strength of the binding interaction, and levels of any nonspecificbinding. Incubation can be for as short at 1 min. or can be for greaterthan 24 hrs. In certain embodiments, incubation overnight.

After incubation, the mixture is washed with buffer to remove unboundtarget molecules. The beads having bound target molecules are thenincubated with the candidate mixture of nucleic acids. The beads havingbound target molecules can be loaded into an HPLC column prior toincubating with the candidate mixture. If the beads having bound targetmolecules are loaded into the HPLC column prior to incubation with thecandidate mixture, incubating of the candidate mixture and the targetmolecule occurs on the column.

Alternatively, the beads having bound target molecule can be incubatedwith the candidate mixture and then the mixture of bead / targetmolecule / nucleic acid complexes and remainder of the candidate mixturecan be loaded into the HPLC column. FIG. 1 shows incubation of thecandidate mixture with the beads having bound target molecules prior toloading into the HPLC column. After incubation is complete, the bead /target molecule / nucleic acid complexes and remainder of the candidatemixture are loaded into the HPLC column. Incubation times can be easilydetermined by one of skill in the art. Factors that influence incubationtime include type of target molecule, the make-up of the candidatemixture, strength of the binding interaction, and levels of anynonspecific binding. Incubation can be for as short at 1 min. or can befor greater than 24 hrs. In certain embodiments, incubation occursovernight.

After the candidate mixture has been incubated with the target moleculesbound to the beads for sufficient time that bead / target molecule /nucleic acid complexes can form, an HPLC elution gradient is applied tothe column in order to obtain the nucleic acid ligands of the targetmolecule. During the elution process the effluent will be enriched innucleic acid ligands of higher affinity for the target molecule, andeventually the final fractions contain the nucleic acid ligands of thehighest affinity to the target molecule (FIG. 1).

The gradient profile typically includes a linearly increasingconcentration of target molecule. The gradient profile also includes anend portion. In certain embodiments, the end portion includes a linearlyincreasing concentration of target molecule. In other embodiments, theend portion includes a linearly increasing concentration of targetmolecule and a chaotropic agent. The unbound nucleic acids and thenucleic acids that have some affinity for the target molecule will elutefrom the column prior to the end portion of the gradient (FIG. 1). Incertain embodiments, the unbound nucleic acid will elute at a beginningportion of the gradient and the nucleic acids that have some affinityfor the target molecule will elute at a middle portion of the gradient.

The nucleic acids with greatest affinity for the target molecule requirea high concentration of target molecule in the effluent to elute fromthe column. The nucleic acids with the greatest affinity for the targetmolecule elute at the end portion of the gradient profile, when theconcentration of target molecule in the effluent is the highest (FIG.1). These nucleic acids of the candidate mixture are identified as thenucleic acid ligands of the target molecule. In certain embodiments,some nucleic acids may show exceptionally high affinity to the targetmolecule and will not be eluted even when the target molecule in theeffluent reaches its maximum. In this embodiment, those very selectivestructures are obtained at the end portion of the elution process usingan increasing concentration of a chaotropic agent, such as urea,guanidinium chloride, SCN⁻, or LiBr. Thus the fractions of the highestaffinity nucleic acid ligands will be eluted gradually.

In other embodiments, the single step separation protocol involvesnative PAGE electrophoresis. In native PAGE, proteins are separatedaccording to the net charge, size and shape of native structure of theprotein. Electrophoretic migration occurs because most proteins carry anet negative charge in alkaline running buffers. The higher the negativecharge density (more charges per molecule mass), the faster a proteinwill migrate. At the same time, the frictional force of the gel matrixcreates a sieving effect, retarding the movement of proteins accordingto size and three-dimensional shape. Small proteins encounter only asmall frictional force while large proteins encounter a largerfrictional force. Thus native PAGE separates proteins based upon bothcharge and mass.

Because no denaturants are used in native PAGE, subunit interactionswithin a multimeric protein are generally retained and information canbe gained about the quaternary structure. In addition, some proteinsretain enzymatic activity following separation by native PAGE. Thus, itmay be used for preparation of purified, active proteins.

In this separation protocol, target molecules are loaded into lanes of agradient gel. An electric current is applied, causing the targetmolecules to migrate to a position in the gel. The gradient gel preventsthe target molecule from migrating to the end of the gel, instead, thetarget molecule is immobilized at a single position in the gel. Eachlane of the gel may contain the same target molecule. Alternatively,each lane of the gel may contain a different target molecule.

Once the target molecule has been immobilized in the gel, the lanes ofthe gel are loaded with the candidate mixture of nucleic acids. Eachlane of the gel may be loaded with the same candidate mixture.Alternatively, each lane of the gel may contain a different candidatemixture. The electric current is applied and the candidate mixturemigrates through the gel, while the target molecule remains immobilizedat its position in the gel. As the candidate mixture migrates to theposition in the gel where the target molecule is immobilized, thenucleic acids of the candidate mixture interact with the targetmolecule. Only the nucleic acids having the highest affinity for thetarget molecule, i.e., the nucleic acids that can withstand effect ofdilution by the running buffer and effect of the electrostatic field,remain bound to the target molecule. The remainder of the candidatemixture, i.e., nucleic acids that have a lesser affinity for the targetmolecule or nucleic acids that have no affinity for the target molecule,will not be able to withstand the forces being applied and will not becapable of remaining bound / binding the target molecule, thus migratingto an end of the gel.

The nucleic acids that remain bound to the target molecule areidentified as the nucleic acid ligands of the target molecule. Thesenucleic acid ligand / target molecule complexes may be cut from the geland application of a chaotropic agent may be used to dissociate thenucleic acid ligands from the target molecules.

The nucleic acid ligands that are obtained by methods of the inventionmay then be sequenced. Any sequencing method known in the art e.g.,ensemble sequencing or single molecule sequencing, may be used. Oneconventional method to perform sequencing is by chain termination andgel separation, as described by Sanger et al., Proc Natl Acad Sci USA,74(12): 5463 67 (1977). Another conventional sequencing method involveschemical degradation of nucleic acid fragments. See, Maxam et al., Proc.Natl. Acad. Sci., 74: 560 564 (1977). Finally, methods have beendeveloped based upon sequencing by hybridization. See, e.g., Drmanac, etal. (Nature Biotech., 16:54-58, 1998). The contents of each of referenceis incorporated by reference herein in its entirety.

In certain embodiments, sequencing is performed by the Sanger sequencingtechnique. Classical Sanger sequencing involves a single-stranded DNAtemplate, a DNA primer, a DNA polymerase, radioactively or fluorescentlylabeled nucleotides, and modified nucleotides that terminate DNA strandelongation. If the label is not attached to the dideoxynucleotideterminator (e.g., labeled primer), or is a monochromatic label (e.g.,radioisotope), then the DNA sample is divided into four separatesequencing reactions, containing four standard deoxynucleotides (dATP,dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is addedonly one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP).These dideoxynucleotides are the chain-terminating nucleotides, lackinga 3′-OH group required for the formation of a phosphodiester bondbetween two nucleotides during DNA strand elongation. If each of thedideoxynucleotides carries a different label, however, (e.g., 4different fluorescent dyes), then all the sequencing reactions can becarried out together without the need for separate reactions.

Incorporation of a dideoxynucleotide into the nascent, i.e., elongating,DNA strand terminates DNA strand extension, resulting in a nested set ofDNA fragments of varying length. Newly synthesized and labeled DNAfragments are denatured, and separated by size using gel electrophoresison a denaturing polyacrylamide-urea gel capable of resolving single-basedifferences in chain length. If each of the four DNA synthesis reactionswas labeled with the same, monochromatic label (e.g., radioisotope),then they are separated in one of four individual, adjacent lanes in thegel, in which each lane in the gel is designated according to thedideoxynucleotide used in the respective reaction, i.e., gel lanes A, T,G, C. If four different labels were utilized, then the reactions can becombined in a single lane on the gel. DNA bands are then visualized byautoradiography or fluorescence, and the DNA sequence can be directlyread from the X-ray film or gel image.

The terminal nucleotide base is identified according to thedideoxynucleotide that was added in the reaction resulting in that bandor its corresponding direct label. The relative positions of thedifferent bands in the gel are then used to read (from shortest tolongest) the DNA sequence as indicated. The Sanger sequencing processcan be automated using a DNA sequencer, such as those commerciallyavailable from PerkinElmer, Beckman Coulter, Life Technologies, andothers.

In other embodiments, sequencing of the nucleic acid is accomplished bya single-molecule sequencing by synthesis technique. Single moleculesequencing is shown for example in Lapidus et al. (U.S. Pat. No.7,169,560), Quake et al. (U.S. Pat. No. 6,818,395), Harris (U.S. Pat.No. 7,282,337), Quake et al. (U.S. patent application number2002/0164629), and Braslaysky, et al., PNAS (USA), 100: 3960-3964(2003), the contents of each of these references is incorporated byreference herein in its entirety. Briefly, a single-stranded nucleicacid (e.g., DNA or cDNA) is hybridized to oligonucleotides attached to asurface of a flow cell. The oligonucleotides may be covalently attachedto the surface or various attachments other than covalent linking asknown to those of ordinary skill in the art may be employed. Moreover,the attachment may be indirect, e.g., via a polymerase directly orindirectly attached to the surface. The surface may be planar orotherwise, and/or may be porous or non-porous, or any other type ofsurface known to those of ordinary skill to be suitable for attachment.The nucleic acid is then sequenced by imaging the polymerase-mediatedaddition of fluorescently-labeled nucleotides incorporated into thegrowing strand surface oligonucleotide, at single molecule resolution.

Other single molecule sequencing techniques involve detection ofpyrophosphate as it is cleaved from incorporation of a single nucleotideinto a nascent strand of DNA, as is shown in Rothberg et al. (U.S. Pat.Nos. 7,335,762, 7,264,929, 7,244,559, and 7,211,390) and Leamon et al.(U.S. Pat. No. 7,323,305), the contents of each of which is incorporatedby reference herein in its entirety.

If only a minimal amount of the nucleic acid ligand is obtained from thecandidate mixture, PCR can be performed on the nucleic acid ligand inorder to obtain a sufficient amount of nucleic acid ligand forsequencing (See e.g., Mullis et al. (U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159) and Saiki, R. K., et al., (Science,239:487-491, 1988), the contents of each of which are incorporated byreference herein in its entirety).

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Identifying Nucleic Acid Ligands using an HPLCGradient Elution Profile

Column Preparation

A SUPELCO ASCENTIS Si HPLC Column, 3 μm particle size, length×I.D. 3cm×2.1 mm was obtained from Sigma-Aldrich (P No 581522-U). The columnstationary phase was modified with aldehyde functionalities. Theprocedure involved:

-   -   Filling the column with 1% ethanol solution of        3-(trimethoxysilyl)butyl aldehyde (United Chemical Technologies,        Bristol, Pa. PNo; PSX1050);    -   Incubation of the filled column for 30 min at room temperature;    -   Equilibration by 5 volumes of with absolute ethanol; and    -   Heating the column at 120° C. for 15 min.        The above procedure results in the formation of a thin coating        of butyl aldehyde functionalities ready for protein attachment.

Binding of Target Molecule to Beads

The activated column was attached to a Waters HPLC system (Milford,Mass.), equilibrated with 50 mM PBS, pH 8.0. The target molecule,recombinant anthrax protective antigen (rPA), was applied to the coatedsurface via column filling with 0.5 mL containing 1 mg solution of rPAin the same buffer supplied with 4 mM sodium cyanoborohydride in fiveconsecutive 0.1-mL injections. The Waters HPLC system consisted ofWaters 600E controller and pump, Waters 717plus Autosampler andinjector, and a Waters 996 Photodiode Array Detector. A FRAC-100Fraction Collector (Pharmacia) was used to collect fractions containingnucleic acids.

After the overnight incubation, the column was extensively washed withfresh buffer to remove unbound protein, thus achieving a stable baseline(A₂₈₀<0.001). Binding of 46% of the applied rPA was confirmed. Thisamount of the protein, 460 μg, is equal to 5.55 nmol based on the rPAmolecular weight of 83,000 Da.

The unreacted aldehyde surface groups were subsequently passivated byreaction with ethanolamine under similar conditions. The column waswashed again with fresh 50 mM PBS, pH 8.0 and used for separation of DNArandom 70 mers.

Preparation of Candidate Mixture

A 95.6 nmol batch of random 70 mer (Sigma-Genosys, Woodlands, Tex.) wasreconstituted in 1 mL of 50 mM PBS, pH 8.0. Based on oligomerconcentration, a 58-μL aliquot of this solution would contain 5.55 nmolof the oligonucleotides, which would be necessary to react in theequimolar ratio to the rPA protein bound within the column.

Nucleic Acid Ligand Capture

Narrow I.D. polypropylene tubing was connected directly to the HPLC pumpand immersed into the oligomer solution. This solution was applied at a0.2 mL/min flow rate directly on the silica HPLC column having the rPAand pre-equilibrated with 50 mM PBS, pH 8.0. The intake process wasvisually monitored. The flow rate was stopped when the last portions ofthe DNA oligomer solution reached the pump intake valve. Based on thegeometrical estimation of the column void volume as being 52 μL (˜50%porosity) by this procedure the column was filled with the amount ofoligimers that was close to 5.55 nmol, or equimolar to the amount ofrPA.

The column was left in the HPLC system to achieve nucleic acid ligandbinding to the potential target molecule, rPA, on the surface ofstationary phase. After 3 hrs, the 0.2 mL/min flow of the 50 mM PBS, pH8.0 was resumed. The wash was continued until the absorbance at 260 nmreached less than 0.001 values.

Nucleic Acid Ligand Elution

The nucleic acid ligand elution was performed in one step using agradient profile. The gradient profile involved an increasing gradientof the rPA concentration in the mobile phase. The end portion of thegradient profile involved an increasing gradient of the rPAconcentration and an increasing gradient of a chaotropic agent, sodiumthiocyanate (NaSCN). In order to reduce the total amounts of therequired rPA, a direct HPLC pump feed method was used. A syringe pumpwas used to generate the gradient of the eluent concentration bycontinuously delivering a concentrated solution of rPA and/or NaSCN intoa vial shaken on a vortex and containing measured amount of theequilibration buffer. The mixed eluent was applied on the column at 0.2mL/min rate by the HPLC pump connected directly to the mixing vial. The0.1 mL fractions were collected and dialyzed against di-water in 1250 Dacut off tubing to remove buffer salts. The dialysis step was importantto remove NaSCN capable of absorbing UV light at 260 nm.

In order to confirm the elution of DNA oligonucleotides from the column,the A₂₆₀ and A₂₈₀ UV absorbance of the fractions were used. FIG. 2depicts the ratio of A₂₆₀/A₂₈₀ as a function of the eluted volume. Aslong as the A₂₆₀/A₂₈₀ ratio for the DNA oligomers greatly exceeds 1.00and the same ratio for the proteins is smaller than 1, this parameterallows detecting fractions enriched with the DNA oligomers. The UVspectra of the random 70 mer and rPA are shown in FIG. 2.

The data show that the ratio varied from 0.8 to 1.6 during the gradientprofile. A substantial number of fractions showed ratios of more than1.00 (highest was equal to 1.57 and eluted at 13.0 to 13.5 mL). Whilefractions obtained during elution with the rPA gradient containedprotein and showed the maximum A₂₆₀/A₂₈₀ of 1.20, the fractions elutedwith NaSCN were absorbing more intensively at 260 NM than at 280 nm, andthe 13.0 to 13.5 mL fraction had the ratio equal to that of the used DNAoligomers (FIG. 3).

Nucleic Acid Ligand Identification and Affinity

Following the above described procedure a nucleic acid ligand wasisolated from the last HPLC fraction. The nucleic acid ligand wassequenced at Sequagen Inc. (Worchester, Mass.), the sequence of which isprovided below:

(SEQ ID NO: 1) TACGACTCACTATAGGGATCCCGAGCTGCAATGGAGTGATTACTAGGGATGTGCGAGGCGCCGAATTCCCTTTAGTGAGGGTT.This nucleic acid ligand showed an affinity to rPA of at least pMlevels.

The affinity of this structure to rPA was estimated based onquantification of rPA detected by the biotinilated nucleic acid ligandduring an ELISA process in a 96-well micro-titer plate format. Morespecifically, the ALISA (Aptamer-Linked Immobilized Sorbent Assay)procedure described in Vivekananda et al. (Lab Invest. 86(6):610-618,2006) was adapted. The procedure included the following main steps:

-   -   The serial dilutions of rPA stock solution were used to coat the        micro-titer plate wells in a concentration of 0.08 to 5.4 μg        (approximately 1.0 to 68 pmol, assuming rPA MW of ˜80,000 g/mol)        per well.    -   After exposure, the non-specific binding sites were blocked with        BSA solution.    -   The biotinilated nucleic acid ligand (synthesized at Sigma        Genosys) solution was added (16 pmol/well). The nucleic acid        ligand / biotin conjugate had the following structure:

(SEQ ID NO: 2) [Btn]TACGACTCACTATAGGGATCCCGAGCTGCAATGGAGTGATTACTAGGGATGTGCGAGGCGCCGAATTCCCTTTAGTGAGGGTT

-   -   After exposure the wells were washed and supplied with the        solutions of polymerized streptavidin-HRP conjugates (Sigma        Aldrich), washed again and supplied with hydrogen peroxide        solution of the ABTS substrate.    -   The rPA was eventually determined using readings of OD rate at        405 nm during 10 to 30 min of the color development reaction.

The absorbance rates were measured and graphed. The blank absorbancerates (control, no rPA) was subtracted from the absorbance of thecorresponding well contained rPA. The sequential repeats were performedto confirm the consistency of the acquired data. The absorbance rate asa function of the rPA amount is depicted in FIG. 3 panels A and B. Dataherein confirm the affinity of the nucleic acid ligand isolated bymethods of the invention to rPA of at least pM levels.

Example 2 Identifying Nucleic Acid Ligands using Gel Electrophoresis

Preparation of Candidate Mixture

DNA oligomers were obtained from a well established library of PCRamplifiable DNA randomers described in Vivekananda et al. (Lab Invest.86(6):610-618, 2006). This library was custom manufactured atSigma-Genosys (The Woodlands, Tex.).

Nucleic Acid Ligand Capture

A solution containing infectious prion protein that resulted in chronicwaste disease (PrP^(CWD)) was loaded into each well of a gradient gel.Native PAGE electrophoresis was performed to immobilize the infectiousprion at a position in the gel. After immobilization of the infectiousprion at a position in the gel, the concentrated solution of DNArandomers was loaded into each well of the gel. Electrophoresis wasperformed for a second time so that the DNA randomers would migratethrough the gel already having the infectious prions immobilized at aposition in the gel. Per each well, 25 μL of the prion protein solutionand 25 μL of DNA oligomer solution containing 3.9 μg DNA weresequentially applied and electrophoretically resolved.

During the second electrophoretic step, the PrP^(CWD) proteins barelymoved in the electrophoretic field and stayed trapped in the network ofthe gradient gel, while the small DNA oligomers (MW of 21 kDa) were ableto migrate towards the positive electrode. Only those DNA oligomers thatshowed high affinity to PrP^(CWD) and could withstand the effect ofdilution by the running buffer and the effect of the electrostatic fieldremained attached to the target protein. Remaining DNA randomersmigrated past the PrP^(CWD) to an end of the gel.

After the second electrophoretic step, the gels were stained withEthidium Bromide (EB) to detect and quantify DNA oligomer bound to thePrP^(CWD) protein within the gel. After performing the above procedureretention of some small quantities of DNA oligonucleotides located onthe PrP^(CWD) entrapped in the gel was observed (FIG. 4). FIG. 4A is acontrol showing an electrophoresis run of only DNA randomers, noprotein. FIG. 4B shows gels obtained by sequential application andelectrophoresis of protein and DNA randomer solutions. The area of DNArandomer/PrP^(CWD) complexes stained with EB due to presence of DNA iscircled.

As shown in FIG. 4B, sub ng quantities of DNA were observed in the areaof localization of DNA randomers complexed with PrP^(CWD) protein. Thisallowed collection and amplification/sequencing of this DNA fraction asa potential candidate for PrP^(CWD) detection. The estimate of theaffinity of the retained aptamer was provided using the estimatedamounts of PrP^(CWD) (˜400 ng, FIG. 4) and nucleic acid ligand (˜0.18ng, FIG. 5), and average molecular weight of the PrP^(CWD) protein of275×10³ g/mol. Thus, the dissociation constant was determined byEquation 2.K _(d) =[PrP ^(CWD)]×[Apt]/[PrP ^(CWD)Apt]/MWPr ^(CWD)protein,  Equation 2where: [PrP^(CWD)] is the equilibrium quantity of the prion protein(ng); [Apt] is the equilibrium quantity of the retained aptamer (ng);[PrP^(CWD) Apt] is the is the equilibrium quantity of the complex of theprion protein and retained aptamer (ng); and MWPrP^(CWD)protein is themolecular weight of the detected MWPrP^(CWD)protien ng/nmol.K _(d)=400×0.18/418/(275×10³);K _(d)=6×10 ⁻⁵ nmol

Nucleic Acid Ligand Isolation

In order to obtain protein free aptamers via PAGE, the zones containingnucleic acid ligands bound to PrP^(CWD) were carefully cut from thepolyacrylamide gels and treated with chaotropic agent (2.0 M solution ofsodium thiocyanate, NaSCN). A total of three 1×8 cm gel fragments wereimmersed in 10 mL of the NaSCN solution in a 50 mL Falcon tube andvortexed for 1 hr at ˜1000 rpm. The resultant gel-water suspension wasfiltered through a 0.8 micron cellulose acetate syringe filter. Thefiltrate was analyzed for the presence of DNA aptamers by PAGE (4-20%gradient gel) with silver staining according to the protocol provided byBioRad Laboratories. DNA 70-randomer was used to serve as standards forthis run. As seen in FIG. 5, the DNA nucleic acid ligands were dilutedgiven the faint bands appearing in the gel.

Nucleic Acid Ligand Amplification

The nucleic acid ligands were amplified by PCR in a G-storm thermalcycler at IST. More specifically, PCR amplification was performed byadding the collected nucleic acid ligands, primers, and nuclease freewater to the EconoTaq 2× Master Mix (Table 1) and followingamplification steps depicted in Table 2.

TABLE 1 PCR reagent composition Amount Final Reagent (μl) ConcentrationConcentration Lucigen Econotaq 2X 25 2X 1X Master Mix Primer AP7: 1.0 50pmol/μl 1 pmol/μl TAC GAC TCA CTA TAG GGA TCC (SEQ ID NO: 3) Primer AP3:1.0 50 pmol/μl 1 pmol/μl AAC CCT CAC TAA AGG GAA TT (SEQ ID NO: 4)Nucleic acid ligand: 1.0 ~1 ng/μl <1 ng/μl 5′-TAC GAC TCA CTA TAG GGATCC-(N = 28)-GAA TTC CCT TTA GTG AGG GTT-3′ (SEQ ID NO: 5) Nuclease FreeWater 22.0 — —

TABLE 2 PCR steps Cycling step Temperature (° C.) Time # of CyclesInitial Denaturation 95  2 min 1 Denaturation 95 30 sec 30 Annealing*50-65 *52.9 30 sec 30 Extension 72 30 sec 30 Final Extension 72  5 min 1Hold  4 overnight 1 *Optimum annealing temperatureIn this manner 4.0 mL solution containing 100 μg of >80% pure nucleicacid ligands was generated (FIG. 6, Lane B).

Nucleic Acid Ligand Identification

The PCR amplification provided amplified material that was sequenced atSequegen Inc, Worcester, Mass. The obtained nucleic acid ligandsequences were used to generate micromole quantities of the purifiednucleic acid ligand sequences at Sigma-Genosys (The Woodlands, Tex.).The sequences of the obtained nucleic acid ligands were as follows:

(SEQ ID NO: 6) TACGACTCACTATAGGGATCCGTTTTTCCGTACTTCTTAAATCGAATTCCCTTTAGTGAGGGTT; (SEQ ID NO: 7)TACGACTCACTATAGGGATCCTTCTCCGCACTACTTTACCTCGAGTGCT ATTCCCTTTAGTGAGGGTT.

Nucleic Acid Ligand Affinity and Specificity Assay

To evaluate the specificity of the generated DNA nucleic acid ligands,quantitative real-time PCR (qrt-PCR) was used to quantify low amounts ofbound nucleic acid ligand from PrP^(CWD) positive biological samples.The following process was used:

-   -   1. Diluted the elk strain of CWD prions (designated as E2) used        in the nucleic acid ligand isolation protocol in PBS from 1:10        to 1:100,000.    -   2. 1:10 dilutions of uninfected normal brain homogenate (NBH)        were used as negative controls.    -   3. 20 μL of each dilution sample was incubated with 180 μL of 1        μM of two different nucleic acid ligand (SEQ ID NO: 6 and SEQ ID        NO: 7) for twenty minutes at room temperature.    -   4. Unbound nucleic acid ligands were removed from the samples by        dilution with 300 μL PBS and filtration through a 100 Kd spin        column.    -   5. Step 4 was repeated twice, substituting 500 μL 500 mM NaSCN        for PBS.    -   6. Samples were concentrated to fifty microliters, and nucleic        acid ligands purified using the DNeasy blood and tissue kit        (Qiagen).    -   7. Nucleic acid ligands were eluted from columns with 50 μL        Tris-EDTA buffer and ten microliters used for nucleic acid        ligand detection by real-time PCR using primers specific for        nucleic acid ligands and SYBR Green fluorescent        DNA-intercalating dye.    -   8. Two HOH-only samples were used as negative controls for PCR.    -   9. All samples were incubated for one pre-melt cycle for three        minutes at 95° C., then thirty cycles of twenty seconds at        95° C. and 45 seconds at 50° C. for annealing and extension and        a final cycle for five minutes at 50° C.

After forty PCR cycles, both negative HOH controls (cyan and greentraces (FIG. 7) and Table 3) remained below fluorescent detectionthreshold (orange horizontal line). The 1:10 E2 dilutions were off thescale where the fluorescence signal was detected without the need foramplification (C_(T)=0). All other E2 dilutions were detected with C_(T)values of 11.7 for 1:100 to 20.3 for 1:100 000 dilutions. Backgrounddetection of nucleic acid ligands eluting with the NBH dilutions (redand blue traces, FIG. 7) was easily distinguished from all othersamples, which were well above 1000 relative fluorescence units. Table 3summarizes the results shown in FIG. 7.

TABLE 3 C_(T) values for RT-PCR samples Well Identifier Ct A01 HOH N/AA02 HOH N/A B01 1:10 E2 (SEQ ID NO: 6) 0 B02 1:10 E2 (SEQ ID NO: 7) 0C01 1:100 E2 (SEQ ID NO: 6) 11.7 C02 1:100 E2 (SEQ ID NO: 7) 11.4 D011:1000 E2 (SEQ ID NO: 6) 11.4 D02 1:1000 E2 (SEQ ID NO: 7) 12.3 E011:10,000 E2 (SEQ ID NO: 6) 13.3 E02 1:10,000 E2 (SEQ ID NO: 7) 15.7 F011:100,000 E2 (SEQ ID NO: 6) 20.3 F02 1:100,000 E2 (SEQ ID NO: 7) 16.4G01 1:10 NBH (SEQ ID NO: 6) 17.4 G02 1:10 NBH (SEQ ID NO: 7) 28.4

In Table 3, the C_(T) values represented the number of cycles requiredfor the fluorescence signal to cross the set threshold. A C_(T) value≦29 indicated an abundance of the target, a C_(T) value of 30-37indicated a positive detection of the target, and a C_(T) value of 38-40indicated the presence of a weak or small amount of the target.

This data show that the two nucleic acid ligands identified by methodsof the invention successfully detected up to a 100,000-fold dilution ofE2 CWD prions. This detection threshold is at least 100-fold greaterthan detection by conventional western blotting. Further advances inoptimization of the assay to achieve a detection limit to 1,000,000dilutions of the prion sample are described below.

To simulate the real low pg and fg concentrations of infectious prionsin bodily fluids, the affinity of the DNA nucleic acid ligands wasfurther evaluated at 1:100,000 dilutions of brain homogenate (actualworking dilution of 1.25×10⁶ under the established PCR protocol). Thisdilution level represents a target sample concentration 0.3 pg/mL ofPrP^(Sc) in the tested brain homogenate which is well within the rangeof infectious prion levels found in the blood of live animals (Brown etal., J Lab Clin Med, 137(1):5-13, 2001). Therefore, this sensitivityprovides for tests to be conducted on bio-fluids collected from liveanimals. For example, the concentration of the infectious prions in theblood of infected animals can be estimated at sub pg/mL levels, whilethe threshold of sensitivity for the detection of infectious prionsextracted from scrapie-infected hamster brain has been estimated at 5pg/mL (Brown et al., J Lab Clin Med, 137(1):5-13, 2001). The estimatedconcentration of PrP^(Sc) in the urine of infected test animals has beenestimated in the fg/mL (0.001 pg) levels (Gonzalez-Romero et al., FEBSLett, 582(21-22):3161-3166, 2008). Therefore, the DNA nucleic acidligands developed above can be used to detect infectious prions in about10 μL of blood or 1 mL of urine.

Data obtained at the 1,000,000 dilutions showed that the infectiousprions were detected (total 1.25 x 10⁶ dilution) of the tested specimen.This corresponds to significant improvement of the detection limit to0.025 pg/mL or 250 atto-grams in a 10 μL sample.

The detection of infectious prions in the 1:100,000 dilutions (C_(T) of16.4-20.3) are comparable to the low-level detection of nucleic acidligands in the NBH samples (blue and red traces, C_(T) of 17.4-28.4)which were at 1:10 dilutions. In addition, the relative fluorescentsignals of the NBH samples were much lower and easily distinguishablefrom test samples (see FIG. 7). These data also showed that optimizationremoves the low-level detection of the NBH sample altogether. Thefluorescence signal of the NBH samples at 1:100 dilutions would beextremely weak to non-existent.

To completely eliminate background nucleic acid ligand noise in thesamples, the samples were further treated with ten units of DNAse I forone minute to remove unbound nucleic acid ligands, while PrP^(CWD)-boundnucleic acid ligands were protected from digestion. Samples were thentreated with 50 μg/mL proteinase K (PK) to eliminate DNAse I activity,then heat inactivated PK for ten minutes at 95° C. to maintain integrityand activity of the Taq polymerase used in the RT-PCR amplification ofbound nucleic acid ligands. This DNAse I/PK/heat inactivation protocolcompletely eliminated possible background amplification of nucleic acidligands in our negative control samples (FIG. 8) and obviated the neednot only for NaSCN treatment and filtration, but also for nucleic acidligand purification using the DNEasy tissue kit. The DNAse I and PKtreatment greatly decreased the organic complexity of the samples, whichcould be used directly in the QRT-PCR reaction without the time andexpense of further DNA extraction. The entire assay was completed fromnucleic acid ligand incubation to RT-PCR to data analysis, in less thanthree hours. CWD prions were successfully detected in a 10⁻⁶ dilution ofbrain homogenate from a CWD-infected elk, corresponding to athousand-fold increase in sensitivity over conventional proteinase Kdigestion/western blotting. Data also show prion detection in archivedspleen tissue from mice infected with CWD (Table 4).

TABLE 4 Summary of additional data generated from subsequent bindingassays Sample Prion¹ Dilution² Detection³ Brain Spiked 10⁻² + BrainSpiked 10⁻³ + Brain Spiked 10⁻⁴ + Brain Spiked 10⁻⁵ + Brain Spiked10⁻⁶ + Brain Infected 10⁻³ + Brain Negative 10⁻¹ − Brain Negative 10⁻² −Brain Negative 10⁻³ − Spleen Infected 10⁻¹ + Spleen Negative 10⁻¹ −

Generation of the specific nucleic acid ligand PCR products wereconfirmed by both melt curve analysis (FIG. 9A) and agarose gelelectrophoresis (FIG. 9B) of amplified samples.

Data herein show that the selected DNA nucleic acid ligands generated bymethods of the invention could detect infectious prions at levels as lowas 0.03 pg/mL concentrations directly in 20 μL samples of biologicalspecimens. This sensitivity is at least 1000-fold higher than what isachievable in immuno-enzymatic assays. Data herein further show thespecificity of these DNA nucleic acid ligands for infectious prions overnormal prions. There were no false positive or negative reactions incontrols containing normal prion protein or no prion protein.

These results show that DNA nucleic acid ligands generated by methods ofthe invention can detect very low concentrations of infectious prionthat are representative of concentrations found in biological fluids orsamples such as blood, urine and feces.

What is claimed is:
 1. A method for identifying a nucleic acid ligand ofa target molecule from a candidate mixture of nucleic acids, the methodcomprising: contacting at least one target molecule with a solutioncomprising a candidate mixture of nucleic acids, wherein the nucleicacids have different affinities for the target molecule; and separatingin a single step nucleic acids that bind the target molecule withgreatest affinity from nucleic acids that bind the target molecule witha lesser affinity and nucleic acids that do not bind the targetmolecule, wherein separating is by a liquid chromatography or geltechnique using a gradient profile that comprises a linearly increasingconcentration of the target molecule and the end portion of the gradientprofile comprises a linearly increasing concentration of the targetmolecule and a linearly increasing concentration of a chaotropic agent,and wherein the separating step is performed only a single time, therebyidentifying the nucleic acid ligand to the target molecule.
 2. Themethod according to claim 1, further comprising sequencing the nucleicacid ligand.
 3. The method according to claim 2, wherein sequencing is asingle-molecule sequencing by synthesis technique.
 4. The methodaccording to claim 1, wherein the target molecule is selected from thegroup consisting of: a protein or portion thereof, an enzyme, a peptide,an enzyme inhibitor, a hormone, a carbohydrate, a glycoprotein, a lipid,a phospholipid, and a nucleic acid.
 5. The method according to claim 1,wherein the nucleic acid ligand comprises DNA or RNA.
 6. The methodaccording to claim 1, wherein the nucleic acid ligand is singlestranded.
 7. The method according to claim 1, wherein the nucleic acidligand is double stranded.